Abstract
Mutations in glucocerebrosidase (GBA) are a common risk factor for Parkinson's disease (PD). The scavenger receptor class B member 2 (SCARB2) gene encodes a receptor responsible for the transport of glucocerebrosidase (GCase) to the lysosome. Two common SNPs in linkage disequilibrium with SCARB2, rs6812193 and rs6825004, have been associated with PD and Lewy Body Disease in genome wide association studies. Whether these SNPs are associated with altered glucocerebrosidase enzymatic activity is unknown.
Our objective was to determine whether SCARB2 SNPs are associated with PD and with reduced GCase activity.
The GBA gene was fully sequenced, and the LRRK2 G2019S and SCARB2 rs6812193 and rs6825004 SNPs were genotyped in 548 PD patients and 272 controls. GCase activity in dried blood spots was measured by tandem mass spectrometry. We tested the association between SCARB2 genotypes and PD risk in regression models adjusted for gender, age, and LRRK2 G2019S and GBA mutation status. We compared GCase activity between participants with different genotypes at rs6812193 and rs6825004.
Genotype at rs6812193 was associated with PD status. PD cases were less likely to carry the T allele than the C allele (OR=0.71; p=0.004), but GCase enzymatic activity was similar across rs6812193 genotypes (C/C: 11.88 μmol/l/h; C/T: 11.80 μmol/l/h; T/T: 12.02 μmol/l/h; p=0.867). Genotype at rs6825004 was not associated with either PD status or GCase activity.
In conclusion, our results support an association between SCARB2 genotype at rs6812193 and PD, but suggest that the increased risk is not mediated by GCase activity.
Introduction
Heterozygous mutations and variants in glucocerebrosidase (GBA) are present in 3-5% of individuals with Parkinson's disease (PD).1,2 The scavenger receptor class B member 2 (SCARB2) gene encodes a protein, lysosome membrane protein 2 (LIMP-2), that transports β-glucocerebrosidase (GCase) from the endoplasmic reticulum through the Golgi apparatus and endosomes to the lysosome.3 Homozygous mutations in SCARB2 cause a rare form of progressive myoclonic epilepsy, action myoclonus-renal failure (AMRF).3 Affected patients have significantly reduced GCase activity (7 nmol/mg protein/h compared with 15 nmol/mg protein/h in controls),3,4 that is in the range observed for carriers of Gaucher disease (GBA heterozygotes). Two common single nucleotide polymorphisms (SNPs), rs68121935,6 (5′ of SCARB2) and rs68250047,8 (intron in SCARB2), have been shown to be associated with PD5-7,9 and Dementia with Lewy Bodies (DLB)8 in several genetic studies, including large genome-wide association studies. Furthermore, given that homozygous mutations in SCARB2 are associated with reduced GCase activity,3 it is possible that these SNPs modify the risk for PD via modulation of GCase activity. In this study, we tested the hypotheses that: 1) these two SNPs would be associated with PD in a New York PD cohort; and 2) that protective variants are associated with higher GCase enzymatic activity as measured in dried blood spots when compared to carriers of the non-protective variant (e.g., if indeed rs6812193 is associated with lower PD risk via SCARB2 then carriers of the protective nucleotide, T, will have higher GCase activity than carriers of the C allele).
Results
Eight hundred and twenty participants were genotyped for the SCARB2 SNPs, including 548 PD cases and 272 controls. Demographics and GBA and LRRK2 mutation status are presented in Table 1. The genotype and allele frequencies of rs6812193 and rs6825004 are presented in Table 2. Genotype at rs6825004 was not associated with PD status in our cohort, nor was it associated with GCase activity (C/C genotype 11.75 μmol/l/h; C/G genotype 11.94 μmol/l/h; G/G genotype: 11.99 μmol/l/h; p=0.703. This comparison included PD cases and controls and excluded GBA and LRRK2 p.G2019S mutation carriers). Genotype at rs6812193 was associated with PD status. The C allele was associated with a higher risk of PD (Table 2; p=0.004) consistent with previous reports.5,6 However, rs6812193 genotype was not associated with differential GCase activity (C/C: 11.88 μmol/l/h; C/T: 11.80 μmol/l/h; T/T: 12.02 μmol/l/h; p=0.867. This comparison included PD cases and controls and excluded GBA and LRRK2 p.G2019S mutation carriers).
Table 1. Demographics, GBA and LRRK2 mutation status in PD cases and controls.
| PD cases (n=548) | Controls (n=272) | p-value | |
|---|---|---|---|
| Mean age in years, (SD) | 66.0 (10.5) | 65.3 (9.5) | 0.339 |
| Percent male, (n) | 64.4% (353) | 34.9% (95) | <0.001 |
| Percent with at least one Ashkenazi Jewish grandparent, (n) | 43.8% (240) | 39.9% (107) | 0.521 |
| Percent with family history of PD in first-degree relative, (n) | 17.5% (96) | 4.8% (13) | <0.001 |
| Carriers of LRRK2 p.G2019S | 7.3% (40) | 0.7% (2) | <0.001 |
| Carriers of any GBA variants1 | 16.6% (91) | 6.6% (18) | <0.001 |
| Education in years, (SD) | 16.6 (2.9) | 16.7 (2.7) | 0.691 |
| UPDRS- part III, (SD) | 17.9 (10.6) | 1.0 (1.8) | <0.001 |
| MoCA, (SD) | 25.3 (3.7) | 27.0 (2.2) | <0.001 |
| Mean PD age-at-onset, (SD) | 59.2 (11.6) | ||
| Levodopa equivalent daily dose in mg, (SD) | 535 (461) |
Including heterozygotes, homozygotes and compound heterozygotes of GBA mutations and variants11
UPDRS: Unified Parkinson's Disease Rating Scale
MoCA: Montreal Cognitive Assessment
Table 2. Association between rs6812193 and rs6825004 genotypes and Parkinson's disease.
| Status | Controls (n=272) | PD Cases (n=548) | p-value for PD risk | OR for PD (95% CI)1 | |||
|---|---|---|---|---|---|---|---|
| N (%) | GCase enzymatic activity2 | N (%) | GCase enzymatic activity2 | ||||
| μmol/l/h (SD) | μmol/l/h (SD) | ||||||
| rs6812193 | C/C | 115 (42.3%) | 11.85 (3.40) | 273 (49.8%) | 11.82 (3.16) | 0.057 | reference |
| C/T | 123 (45.2%) | 11.77 (3.07) | 229 (41.8%) | 11.77 (3.09) | 0.52 (0.30-0.89); p=0.018 | ||
| T/T | 34 (12.5%) | 12.10 (4.57) | 46 (8.4%) | 11.98 (3.10) | 0.62 (0.36-1.07); p=0.086 | ||
| C | 353 (64.8%) | 775 (70.7%) | 0.0175 | 0.71 (0.56-0.90); p=0.0043 | |||
| T | 191 (35.2%) | 321 (29.3%) | |||||
| rs68250044 | C/C | 125 (46.1%) | 11.94 (3.35) | 243 (44.4%) | 11.63 (2.88) | 0.562 | reference |
| C/G | 115 (42.4%) | 11.90 (3.22) | 229 (41.9%) | 11.97 (3.42) | 1.03 (0.74-1.45); p=0.833 | ||
| G/G | 31 (11.4%) | 12.42 (3.33) | 75 (13.7%) | 11.76 (3.04) | 0.79 (0.48-1.30); p=0.788 | ||
| C | 365 (67.3%) | 715 (65.3%) | 0.438 | 1.17 (0.93-1.47); p=0.1823 | |||
| G | 177 (32.7%) | 379 (34.7%) | |||||
OR=odds ratio; calculated in models adjusted for age, gender, LRRK2 and GBA mutation status.
There was no difference in GCase activity among the genotypes, either in controls or in PD cases. Values presented here after excluding GBA and LRRK2 p.G2019S carriers.
Model included sex, age, rs6812193 genotype and rs6825004 genotype.
rs6825004 was missing on one PD and one control participant.
Discussion
Our findings support previous reports that the genotype at rs6812193 SNP is associated with PD. This SNP, which is in linkage disequilibrium with the SCARB2 gene, was found in association with PD in two large multi-ethnic genome-wide association studies (OR=0.84; p=7.6 ×10-10 and OR=0.907; p=2.95×10 -11) 5,9 and in a German association study (OR=0.86; p=0.02).6 However, the association was not replicated in a Chinese10 or a Greek cohort,11 likely due to either population differences or insufficient power. Here, we confirm the association in a clinic based New York cohort, which consists of 40% AJ PD patients. An association between the SCARB2 gene and PD was hypothesized to be due to differential trafficking of GCase to the lysosome.4 In the current study we hypothesized that if indeed rs6812193 is associated with PD risk via SCARB2, then carriers of the protective nucleotide (T) should have higher GCase activity than carriers of the PD susceptibility allele (C). We have previously shown in dried blood spots that heterozygous GBA carriers have lower GCase activity than non-carriers (by ∼33%), and that idiopathic PD cases have slightly lower GCase activity (by ∼5%) than controls.12 Our current findings do not support the link between rs6812193 and PD through a mechanism of reduced GCase activity (at least not as measured in dried blood spots). There are several possible explanations for our findings. First, rs6812193 is associated with PD via a different gene/mechanism not linked to SCARB2 or GCase. This explanation is supported by a prior study showing that variants in this SNP do not correlate with LIMP-2 mRNA or protein levels in leukocytes from a small sample of controls.13 Second, rs6812193 is associated with PD through SCARB2, but its biological effect is not mediated by GCase activity. Finally, rs6812193 is associated with PD through SCARB2 and GCase pathways but this effect is either present in selected tissues (e.g., brain) and not leukocytes, or it involves altered intracellular routing and localization of GCase that is not apparent in the DBS assay.
In addition, we were not able to replicate the association between rs6825004 and PD. This association was reported in a Greek cohort7 and was also reported in a Lewy Body Disease association study, but not in other PD genome-wide studies.8 It is possible that we did not observe the association because our cohort did not include Dementia with Lewy Body cases.
In summary, we provide evidence that SCARB2 is associated with PD although the mechanism remains unknown. Full sequencing of SCARB2 in PD cohorts and correlation of PD risk with GCase activity may help to clarify these associations further.
Methods and Participants
Participants and Clinical Evaluation
Participants in the ‘SPOT’ study included PD patients and genetically unrelated controls (mostly spouses) from the Center for Parkinson's Disease at Columbia University Medical Center in New York, NY, recruited between 2010-2014.14 The cohort has been previously described.12 In brief, a blood sample and data on demographics, medical history, medication, PD family history,15 the Unified Parkinson's Disease Rating Scale (UPDRS) in the “on” state and the Montreal Cognitive Assessment (MoCA)16 were collected from consecutive PD cases, as defined by the United Kingdom PD brain bank criteria (except that we did not exclude cases with a family history of PD).17 A blood sample was collected from genetically unrelated control individuals, mostly spouses. All study procedures were approved by the Columbia University IRB, and all participants signed informed consent.
Genotyping of GBA and SCARB2
All study participants were fully sequenced for GBA mutations and genotyped for the LRRK2 p.G2019S mutation as previously described.12 Two SCARB2 SNPs were genotyped by TaqMan SNP genotyping assays, rs6812193 and rs6825004 (assay IDs: C__31139749_10 and C___1129894_20, respectively) following the manufacturer's instructions, and the genotypes were called using QuantStudio™ 7 Flex Real-Time PCR System and Software (Applied Biosystems).
GCase Activity Assay
Dried blood spots were obtained as previously described.12,18,19 In brief, 75 μl of blood was “spotted” on a filter paper and dried at room temperature for at least 4 hours. GCase activity was measured at Sanofi Genzyme laboratories using a previously published protocol as part of a multiplex assay together with four additional lysosomal enzymes.20Activity was expressed as micromoles of product per liter of whole blood per hour (μmol/l/h). All Sanofi Genzyme scientists were blinded to PD and genetic status.
Statistical Analysis
Demographics, frequency of GBA variants, LRRK2 p.G2019S mutations, rs6812193 and rs6825004 genotypes were compared between PD cases and controls using the Student t test for continuous variables, and the chi-square and Fisher exact tests for categorical variables. We used logistic regression analyses to test whether the SNPs (predictors) are associated with PD status (outcome) in models adjusted for age, gender and GBA and LRRK2 mutation status.
To test the association between GCase activity and the rs6812193 and rs6825004 genotypes, we first compared GCase enzymatic activity by genotypes in the entire sample (including PD cases and controls), and then in PD cases and controls separately. Analyses of GCase activity were repeated excluding all carriers of GBA variants previously associated with reduced GCase activity and the LRRK2 p.G2019S mutation which was previously shown to be associated with increased GCase activity.12
Analyses were performed using SPSS Statistics version 21.0 software (Chicago, IL).
Acknowledgments
Funding for this study provided by the Parkinson's Disease Foundation, the National Institutes of Health (K02NS080915 and UL1 TR000040, formerly the National Center for Research Resources, Grant Number UL1 RR024156) and the Brookdale Foundation. The authors would like to thank Ms. Judy Hull for coordinating the collaboration and Mr. Pauciulo and Dr. Nichols for LRRK2 genotyping.
Potential Conflicts of Interest: Dr. Alcalay is supported by the Parkinson's Disease Foundation, the National Institutes of Health (K02NS080915, and UL1 TR000040, formerly the National Center for Research Resources, Grant Number UL1 RR024156), the Smart Foundation and the Brookdale Foundation. He received consultation fees from Genzyme/Sanofi and Prophase. Ms. Wolf and Drs. Oliva and Zhang are employees of Genzyme/Sanofi. Dr. Fahn reports Consulting and Advisory Board Membership with honoraria: Merz Pharma, Genervon Biotechnology; PixarBio; Lundbeck Pharma. Grants/Research Support: 69Genervon Biotechnology. Dr. Kang reports research grants from NIH, the Michael J Fox Foundation, and the Parkinson Disease Foundation, not related to the submitted work. He consults for Caremark/CVS. Dr. Kuo has received funding from the National Institutes of Health: NINDS #K08 NS083738 (principal investigator), the Louis V. Gerstner Jr. Scholar Award, the Parkinson's Disease Foundation, the American Parkinson's Disease Association, the International Essential Tremor Foundation, NIEHS Pilot Grant ES009089 (principal investigator), and the Smart Foundation. Dr. Marder reports grants from NIH [#NS036630 (PI), 1UL1 RR024156-01(Director PCIR), PO412196- G (Co-I), and PO412196-G (Co-I)], grants from steering committee for U01NS052592, grants from the Parkinson's Disease Foundation, and grants from Michael J Fox Foundation, outside the submitted work.
References
- 1.Sidransky E, Nalls MA, Aasly JO, et al. Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease. N Engl J Med. 2009 Oct 22;361(17):1651–1661. doi: 10.1056/NEJMoa0901281. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Neumann J, Bras J, Deas E, et al. Glucocerebrosidase mutations in clinical and pathologically proven Parkinson's disease. Brain : a journal of neurology. 2009 Jul;132(Pt 7):1783–1794. doi: 10.1093/brain/awp044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Zeigler M, Meiner V, Newman JP, et al. A novel SCARB2 mutation in progressive myoclonus epilepsy indicated by reduced beta-glucocerebrosidase activity. Journal of the neurological sciences. 2014 Apr 15;339(1-2):210–213. doi: 10.1016/j.jns.2014.01.022. [DOI] [PubMed] [Google Scholar]
- 4.Dardis A, Filocamo M, Grossi S, et al. Biochemical and molecular findings in a patient with myoclonic epilepsy due to a mistarget of the beta-glucosidase enzyme. Mol Genet Metab. 2009 Aug;97(4):309–311. doi: 10.1016/j.ymgme.2009.04.011. [DOI] [PubMed] [Google Scholar]
- 5.Do CB, Tung JY, Dorfman E, et al. Web-based genome-wide association study identifies two novel loci and a substantial genetic component for Parkinson's disease. PLoS genetics. 2011 Jun;7(6):e1002141. doi: 10.1371/journal.pgen.1002141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Hopfner F, Schulte EC, Mollenhauer B, et al. The role of SCARB2 as susceptibility factor in Parkinson's disease. Movement Disord. 2013 Apr;28(4):538–540. doi: 10.1002/mds.25349. [DOI] [PubMed] [Google Scholar]
- 7.Michelakakis H, Xiromerisiou G, Dardiotis E, et al. Evidence of an association between the scavenger receptor class B member 2 gene and Parkinson's disease. Movement Disord. 2012 Mar;27(3):400–405. doi: 10.1002/mds.24886. [DOI] [PubMed] [Google Scholar]
- 8.Bras J, Guerreiro R, Darwent L, et al. Genetic analysis implicates APOE, SNCA and suggests lysosomal dysfunction in the etiology of dementia with Lewy bodies. Human molecular genetics. 2014 Dec 1;23(23):6139–6146. doi: 10.1093/hmg/ddu334. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Nalls MA, Pankratz N, Lill CM, et al. Large-scale meta-analysis of genome-wide association data identifies six new risk loci for Parkinson's disease. Nature genetics. 2014 Sep;46(9):989–993. doi: 10.1038/ng.3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Chen S, Zhang Y, Chen W, et al. Association study of SCARB2 rs6812193 polymorphism with Parkinson's disease in Han Chinese. Neuroscience letters. 2012 May 10;516(1):21–23. doi: 10.1016/j.neulet.2012.03.035. [DOI] [PubMed] [Google Scholar]
- 11.Kalinderi K, Bostantjopoulou S, Katsarou Z, Fidani L. Association study of rs6812193 polymorphism with Parkinson's disease in a Greek population. Neuroscience letters. 2013 Apr 29;541:190–192. doi: 10.1016/j.neulet.2013.02.048. [DOI] [PubMed] [Google Scholar]
- 12.Alcalay RN, Levy OA, Waters CC, et al. Glucocerebrosidase activity in Parkinson's disease with and without GBA mutations. Brain : a journal of neurology. 2015 Jun 27; doi: 10.1093/brain/awv179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Maniwang E, Tayebi N, Sidransky E. Is Parkinson disease associated with lysosomal integral membrane protein type-2?: challenges in interpreting association data. Mol Genet Metab. 2013 Apr;108(4):269–271. doi: 10.1016/j.ymgme.2013.01.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Sakanaka K, Waters CH, Levy OA, et al. Knowledge of and interest in genetic results among Parkinson disease patients and caregivers. Journal of genetic counseling. 2014 Feb;23(1):114–120. doi: 10.1007/s10897-013-9618-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Marder K, Levy G, Louis ED, et al. Accuracy of family history data on Parkinson's disease. Neurology. 2003 Jul 8;61(1):18–23. doi: 10.1212/01.wnl.0000074784.35961.c0. [DOI] [PubMed] [Google Scholar]
- 16.Nasreddine ZS, Phillips NA, Bedirian V, et al. The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive impairment. Journal of the American Geriatrics Society. 2005 Apr;53(4):695–699. doi: 10.1111/j.1532-5415.2005.53221.x. [DOI] [PubMed] [Google Scholar]
- 17.Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry. 1992 Mar;55(3):181–184. doi: 10.1136/jnnp.55.3.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Reuser AJ, Verheijen FW, Bali D, et al. The use of dried blood spot samples in the diagnosis of lysosomal storage disorders--current status and perspectives. Mol Genet Metab. 2011 Sep-Oct;104(1-2):144–148. doi: 10.1016/j.ymgme.2011.07.014. [DOI] [PubMed] [Google Scholar]
- 19.Olivova P, Cullen E, Titlow M, et al. An improved high-throughput dried blood spot screening method for Gaucher disease. Clinica chimica acta; international journal of clinical chemistry. 2008 Dec;398(1-2):163–164. doi: 10.1016/j.cca.2008.08.024. [DOI] [PubMed] [Google Scholar]
- 20.Zhang XK, Elbin CS, Chuang WL, et al. Multiplex enzyme assay screening of dried blood spots for lysosomal storage disorders by using tandem mass spectrometry. Clinical chemistry. 2008 Oct;54(10):1725–1728. doi: 10.1373/clinchem.2008.104711. [DOI] [PubMed] [Google Scholar]